A rapid molecular detection method of puccinia coronata based on rpa

Abstract

The application discloses a rapid molecular detection method of Puccinia coronata based on RPA, and belongs to the technical field of agricultural biological disease molecular detection.The application compares and analyzes the genomic sequence difference of Puccinia coronata and other pathogenic bacteria of cereal crops, designs and selects a specific RPA primer pair, and the sequence is shown as SEQ ID NO.1 and SEQ ID NO.2.RPA amplification is carried out by using the primer pair, specific target fragments in the genomic DNA of Puccinia coronata can be rapidly amplified under the condition of constant temperature reaction at 39 DEG C for 15 min, and the size of the amplification product is 223 bp.The detection system established by the application has high specificity and high sensitivity, the detection lower limit reaches 1 pg / micro.L, and early detection of the pathogenic bacteria can be realized at the stage of no symptom in the fifth day after inoculation.The application provides reliable technical support for early rapid diagnosis and field monitoring of Puccinia coronata.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for agricultural biological diseases, specifically to a rapid molecular detection method for oat crown rust based on recombinase polymerase amplification (RPA) technology, which is suitable for early rapid diagnosis of oat crown rust and accurate identification of pathogens. Background Technology

[0002] Oats (Avena sativa L.) belong to the genus Avena of the subfamily Pooideae in the family Poaceae. They are rich in nutrients and have a relatively balanced composition. They not only have high edible value, but are also one of the most important high-quality forage crops in the world.

[0003] Rust is one of the most serious fungal diseases affecting cereal crops, mainly including stripe rust, leaf rust, stem rust, and crown rust. Crown rust is caused by the obligate parasitic fungus *Puccinia coronata* Corda, while oat crown rust is caused by the oat-specific form of this fungus (*Puccinia coronata f. sp. avenae*). It is a typical airborne fungal disease, with its urediniospores able to travel long distances by air currents, causing serious damage to oat production worldwide.

[0004] Oat crown rust primarily infects leaves, but can also spread to leaf sheaths, stems, and ears. In the early stages of infection, scattered or slightly clustered orange-yellow, oval uredinia often form on the leaf surface. After maturation, the epidermis of the uredinia ruptures, releasing a large number of urediniospores, which become the main source of reinfection in the field. In the later stages of growth, black telia, whose epidermis is not easily ruptured, gradually form on diseased parts (especially the underside of leaves). These telia overwinter in a dormant state on diseased plant debris, germinating the following year to produce basidiospores that infect the alternate host, *Rhamnus rubra*, completing the disease cycle.

[0005] This disease significantly damages oat production, causing varying degrees of yield reduction year after year, with particularly severe losses in years of large-scale outbreaks. Simultaneously, pathogen infection significantly reduces grain protein content and germination rate, and degrades the feed quality of straw, thus directly weakening the economic value of oats. Early and accurate identification and rapid diagnosis of the disease are crucial prerequisites for conducting field monitoring, epidemic warning, and precise control. Therefore, establishing accurate and rapid identification techniques for oat crown rust is an urgent need to be addressed to ensure safe oat production.

[0006] Traditional methods for detecting and identifying pathogenic fungi mainly follow the basic principles of Koch's postulates, relying on the observation and description of biological characteristics. While morphological characteristics serve as an important basis for fungal taxonomy and hold an irreplaceable fundamental position, relying solely on these characteristics for pathogen identification not only has limited reliability but also fails to meet the practical requirements of timely and accurate diagnosis in production practice.

[0007] The rapid development of molecular biology techniques has driven a revolutionary change in the field of plant pathogenic fungi detection, effectively compensating for the shortcomings of traditional methods in terms of operational procedures and identification accuracy. Recombinase polymerase amplification (RPA) is a novel multi-enzyme isothermal nucleic acid amplification technique. Its core mechanism relies on the synergistic action of recombinases, single-strand binding proteins, and DNA polymerases with strand displacement capabilities. Under isothermal conditions of 37-42℃, it simulates the in vivo DNA homologous recombination process, achieving rapid exponential amplification of target sequences. Compared with conventional PCR and other techniques, RPA has significant advantages such as rapid reaction, high specificity, low equipment dependence, and good sample tolerance. Detectable amplified products can usually be obtained within 5-20 minutes at 37-42℃, making it considered one of the most promising rapid molecular diagnostic tools.

[0008] Currently, RPA technology is widely used in the detection of bacteria, viruses, fungi, parasites, and drug resistance genes. However, specific RPA detection methods for oat crown rust fungus have not yet been reported. Therefore, establishing a rapid molecular detection method for oat crown rust fungus based on RPA can provide important technical support for achieving efficient and reliable diagnosis of oat crown rust disease. Summary of the Invention

[0009] This invention addresses the shortcomings of existing technologies by providing a rapid molecular detection method for oat crown rust based on RPA. Specifically, this invention compares and analyzes the genomic sequence differences between oat crown rust and other common pathogens of cereal crops, designs and screens specific RPA primers for oat crown rust, and optimizes the system to establish a highly specific and sensitive isothermal rapid molecular detection system for oat crown rust.

[0010] The purpose of this invention is to construct an efficient, sensitive, and easy-to-use molecular detection system for oat crown rust fungus, so as to achieve early diagnosis of the disease and accurate identification of the pathogen, and at the same time provide an effective technical means for disease epidemic monitoring.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] First, using the genomic DNA of *Crassula ovata* as a template, this invention performs PCR amplification using primers EF1-983F (5′-GCYCCYGGHCAYCGTGAYTTYAT-3′) and EF1-1567R (5′-ACHGTRCCRATACCACCSATCTT-3′). The amplified product is cloned and sequenced to obtain a continuous nucleotide sequence containing the target fragment, as shown in SEQ ID NO.3.

[0013] Puccinia coronata f. sp. avenae (oat-specific form of Puccinia coronata f. sp. avenae):

[0014] (SEQ ID NO.3).

[0015] Using the genomic DNA of *Bacillus crown rust* as a template, the same primers were used for amplification and sequencing, and the obtained sequence is shown in SEQ ID NO.4.

[0016] Puccinia coronata var. hordei:

[0017] (SEQ ID NO.4).

[0018] By comparing the sequences of *Oat Crown Rust* with those of *Barley Crown Rust* and other cereal pathogenic fungi, nucleotide regions with high specificity and intraspecific conservation in *Oat Crown Rust* were screened as candidate targets for primer design. Based on this, RPA-specific primer pairs (SEQ ID NO.1 and SEQ ID NO.2) and conventional PCR primer pairs were designed.

[0019] The design of RPA primers strictly adhered to the following principles: primer length was controlled at 30-35 bp, GC content was maintained within the range of 40%-60%, secondary structure formation and primer dimers were avoided as much as possible, high 3′ end stability was ensured, and amplicon length was controlled at 100-300 bp (generally less than 500 bp). Based on these principles, two pairs of candidate RPA primers were designed within the specific target region of *Crassula ovata*. After specificity verification and amplification efficiency comparison, both candidate primer pairs could specifically detect *Crassula ovata*, but primer sets SEQ ID NO.1 and SEQ ID NO.2 showed better band clarity and higher amplification efficiency. Therefore, SEQ ID NO.1 and SEQ ID NO.2 were ultimately selected as the optimal primer pair for the RPA detection system.

[0020] To verify the reliability of the established detection system, this invention conducted specificity analysis and sensitivity tests on the system, and evaluated the actual performance of the rapid molecular detection method for oat crown rust.

[0021] The spread of diseases in the field can often be traced back to early infection and initial lesion formation caused by extremely low levels of primary infectants (even a single pathogenic spore). Based on this, the present invention further designed a simulation experiment of disease caused by infection with a single urediniospore in the laboratory to evaluate the early diagnosis and latent period detection capabilities of the detection system under field infection conditions.

[0022] Specifically as follows:

[0023] First, the present invention provides a rapid molecular detection method for *Crassula capitella* based on recombinase polymerase amplification technology, comprising the following steps:

[0024] (1) Extract genomic DNA from the sample to be tested;

[0025] (2) Using the genomic DNA extracted in step (1) as a template, a recombinase polymerase amplification reaction was carried out using specific primer pairs;

[0026] (3) Detect the amplification products;

[0027] The specific primer pair is:

[0028] Forward primer: 5'-CGATTGTGTCATCGCTTACACTCTGTTGCC-3' (SEQ ID NO.1);

[0029] Reverse primer: 5'-GACGAAGGAAAACCAGGCACTTCTCAACAC-3' (SEQ ID NO.2).

[0030] Preferably, the recombinase polymerase amplification reaction system is as follows: 29.4 μL of buffer A, 2 μL each of forward and reverse primers, 2 μL of template DNA, ddH2O added to a final volume of 47.5 μL, mixed well, and then 2.5 μL of buffer B is added; the concentration of both the forward and reverse primers is 10 μM.

[0031] Preferably, the temperature of the recombinase polymerase amplification reaction is 37-42℃, more preferably 39℃; and the reaction time is 5-30 min, more preferably 15 min.

[0032] Preferably, the detection of amplification products in step (3) is performed by agarose gel electrophoresis.

[0033] Second, this invention provides an RPA primer pair for rapid molecular detection of *Crassula ovata*, wherein the primer pair is:

[0034] Forward primer: 5'-CGATTGTGTCATCGCTTACACTCTGTTGCC-3' (SEQ ID NO.1);

[0035] Reverse primer: 5'-GACGAAGGAAAACCAGGCACTTCTCAACAC-3' (SEQ ID NO.2).

[0036] Preferably, the primer pair amplifies a specific region of the translation elongation factor 1-α gene fragment in the genome of *Crassula ovata*, and the amplification product size is 223 bp.

[0037] Furthermore, the forward primer SEQ ID NO.1 binds to positions 268-297 of SEQ ID NO.3; the reverse primer SEQ ID NO.2 binds to positions 461-490 of SEQ ID NO.3.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) Monitoring and early warning of plant pathogens are important guarantees for effective disease control and agricultural production safety. Although conventional PCR technology has been widely used in this field, its operation usually depends on the laboratory environment and sophisticated thermal cycling equipment, which is difficult to meet the actual needs of rapid detection. Recombinase polymerase amplification (RPA) technology has shown outstanding advantages in the field of nucleic acid amplification due to its simple operation, rapid reaction, high specificity and high sensitivity. Based on the principle of isothermal amplification, this technology breaks through the dependence on thermal cycling equipment and significantly improves the applicability of detection under resource-limited conditions. Based on this, this invention utilizes RPA technology to realize the rapid molecular detection of oat crown rust for the first time.

[0040] (2) The RPA detection system established in this invention is consistent with the results of conventional PCR detection. Both can specifically detect oat crown rust, and the lower limit of its detection sensitivity is 1 pg / μL, showing high detection sensitivity.

[0041] (3) The RPA detection system established in this invention can specifically detect oat crown rust fungus from the genomic DNA of oat seedlings that have not yet shown symptoms after inoculation. This indicates that the system has the ability to detect early diagnosis and latent period, and can provide reliable technical support for the early and rapid diagnosis of oat crown rust in the field. It is of great significance for the early monitoring and early warning of this disease.

[0042] (4) In the RPA primer design process, the present invention sets strict primer screening criteria (length 30-35 bp, GC content 40%-60%, no secondary structure and primer dimer, high 3′ end stability, amplicon length 100-300 bp), and selects the optimal primer pair from 2 pairs of candidate primers by comparing amplification efficiency and band clarity, which further ensures the reliability and stability of the detection system. Attached Figure Description

[0043] Figure 1 The figure shows the agarose gel electrophoresis results for specificity verification of conventional PCR primers in this embodiment of the invention. In the figure, M.600 DNA Marker; 1, 2 *Oat Crown Rust*; 3, *Wheat Stripe Rust*; 4, *Wheat Stem Rust*; 5, *Wheat Leaf Rust*; 6, *Barley Stripe Rust*; 7, *Barley Leaf Rust*; 8, *Barley Crown Rust*; 9, *Oat Stem Rust*; 10, *Barley Sheath Rot*; 11, *Wheat Powdery Mildew*; 12, Uninoculated *Oat Qingyan No. 1* Leaf; 13, Negative Control ddH2O. As shown in the figure, only the reaction system using *Oat Crown Rust* genomic DNA as a template amplified the expected size of the specific target band, while no amplification products were detected in the genomic DNA templates of the other tested strains and the negative control, indicating that the primers used have good specificity.

[0044] Figure 2The figure shows the agarose gel electrophoresis results of the establishment of the RPA detection system and the verification of primer specificity in this embodiment of the invention. In the figure, M.600 DNA Marker; 1, 2 *Crassula capitella* (oat rust); 3, *Stripe rust* (wheat stripe rust); 4, *Stripe rust* (wheat stem rust); 5, *Leaf rust* (wheat leaf rust); 6, *Stripe rust* (barley stripe rust); 7, *Leaf rust* (barley leaf rust); 8, *Crassula capitella* (barley rust); 9, *Stripe rust* (oat rust); 10, *Hymenoplastia spp.* (barley sheath rot pathogen); 11, *Powdery mildew* (wheat powdery mildew); 12, uninoculated *Qingyan No. 1* oat leaf; 13, negative control ddH2O. The results show that only the reaction system using *Crassula capitella* genomic DNA as a template amplified the specific target band, while no amplification products were observed in other tested pathogens and samples from uninoculated oat tissues, consistent with the results of the negative control (ddH2O). This indicates that the RPA detection system has good detection specificity for *Crassula capitella* genomic DNA and can effectively distinguish the target pathogen from other tested wheat pathogenic fungi.

[0045] Figure 3 The agarose gel electrophoresis results for verifying the sensitivity of the RPA detection system established in this invention are shown in the figure. M.600 DNA Marker; 1. 10 ng / μL; 2. 1 ng / μL; 3. 100 pg / μL; 4. 10 pg / μL; 5. 1 pg / μL; 6. 100 fg / μL; 7. 10 fg / μL; 8. 1 fg / μL; 9. Negative control ddH2O. As shown in the figure, the system exhibits a detection limit of 1 pg / μL for *Crassula capitella* genomic DNA, demonstrating high detection sensitivity.

[0046] Figure 4 Optimization of reaction conditions for the *Crassula ovata* RPA system, including: (a) Optimization of reaction temperature; M: 600 bp Marker; lanes 1-5: 33℃, 35℃, 37℃, 39℃, 41℃; lane 6: negative control (ddH2O). (b) Optimization of reaction time; M: 600 bp Marker; lanes 1-6: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min; lane 7: negative control (ddH2O).

[0047] Figure 5The results show the detection results at different infection time points after artificial inoculation of single urediniospores of *Crassula ovata* in oats. In the figure, M: 600 bp Marker; lanes 1-10 represent days 1-10 after inoculation of *Crassula ovata* var. *Qingyan 1*; 11: negative control (ddH2O); 12: leaves of uninoculated *Qingyan 1* var. *A*. (a) shows oat leaf samples at different infection time points (1-10: days 1-10 after inoculation); (b) shows the RPA detection results of the corresponding samples. The results show that specific target amplification bands can be detected as early as day 5 after inoculation, and the band brightness gradually increases with the extension of infection time. These results indicate that healthy oat plants infected with *Crassula ovata* can be effectively detected by the RPA detection system established in this invention during the incubation stage before obvious sporulation symptoms are observed. Detailed Implementation

[0048] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0050] Example 1

[0051] This embodiment provides the establishment of an RPA detection system for oat crown rust, optimization of reaction conditions, and specificity verification experiments.

[0052] 1. Collection, isolation, and purification of the tested oat crown rust fungus and reference strains.

[0053] Oat crown rust specimens were collected from Fengshui Village, Luoyu Town, Xihe County, Longnan City, Gansu Province. Other reference strains were pathogens of common diseases of wheat crops: wheat stripe rust (Puccinia striiformis West. f. sp. tritici Eriks.), wheat stem rust (P. graminis Pers. f. sp. tritici Eriks et Henn.), wheat leaf rust (P. triticina Erikss.), barley stripe rust (P. striiformis West. f. sp. hordei Erikset Henn.), barley leaf rust (P. hordei GH Otth), barley crown rust (P. coronata Corda var. hordei Jin et Steffenson), oat stem rust (P. graminis Pers. f. sp. avenae Eriks et Henn.), and barley sheath rot (Dactylobotrys graminicola SQ He et SR). Wang), wheat powdery mildew [Blumeria graminis (DC.) Speer f. sp. tritici Em. Marchal].

[0054] The oat variety Qingyan No. 1 and the reference strains (wheat variety Mingxian 169 and barley variety Guoluo) were cultivated in a sterile seedling room at 15℃ with a photoperiod of 16 h light / 8 h dark. When the seedlings reached the stage where one leaf was fully expanded, they were used for inoculation. Using a special eyelash inoculation pen, individual urediniospores were isolated and picked under a stereomicroscope and inoculated onto the upper surface of dewaxed leaves. After inoculation, a suitable amount of water mist was sprayed, and each inoculated plant was then covered with a transparent isolation cover and placed in a plastic box with a suitable amount of water at the bottom. The box was then placed in a dark, humidified room at 10℃ and 100% relative humidity for 24-36 hours. After humidification, the plants were transferred to a cultivation greenhouse for further cultivation. After conidia and urediniospore rupture, the inoculum was collected using clean, dry, sterile test tubes to obtain purified inoculum, which was then temporarily stored at 4℃.

[0055] 2. Preparation of DNA templates from *Oat Crown Rust* and reference strains

[0056] Place the collected purified bacterial culture into a 2 mL centrifuge tube, add two 3 mm diameter steel balls, and break the culture using a tissue homogenizer. Then, prepare a DNA template using a fungal genomic DNA extraction kit, following the instructions.

[0057] 3. PCR amplification and purification

[0058] Using the extracted genomic DNA as a template, the primers used for PCR amplification were EF1-983F / EF1-1567R, synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd. The total PCR amplification system was 50 μL, containing 2 μL each of forward and reverse primers (10 μM), 2 μL of template DNA (50 ng / μL), 25 μL of 2×Es Taq MasterMix (Dye) PCR mixture, and 19 μL of ddH2O. The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 35 cycles: 94℃ denaturation for 10 s, 56℃ annealing for 30 s, 72℃ extension for 1 min; after 35 cycles, a final extension was performed at 72℃ for 5 min.

[0059] Primer sequences are: EF1-983F: 5'-GCYCCYGGHCAYCGTGAYTTYAT-3' EF1-1567R: 5'-ACHGTRCCRATACCACCSATCTT-3'

[0060] After amplification, the PCR products were detected by 1% (m / v) agarose gel electrophoresis. Following detection, the products were purified using a gel extraction kit.

[0061] 4. Ligation and transformation of the vector and the target fragment

[0062] The ligation vector was obtained using the pMD™ 18-T Vector Cloning Kit (TaKaRa), and DH5α competent cells were transformed. Positive clones were screened and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0063] 5. Primer design and screening

[0064] The gene sequences of *Crassula capitella* (SEQ ID NO.3) and *Crassula barley* (SEQ ID NO.4), as well as other reference strains, were compared using the sequence alignment software DNAMAN to screen for highly specific and conserved nucleotide regions within *Crassula capitella* as candidate targets for primer design. Sequence alignment confirmed that the primer sequences were compared with corresponding regions in other reference strains (such as *Crassula barley*). The results showed multiple base differences in the binding regions of both the forward and reverse primers. These differing sites ensured that the designed primers could specifically bind to the target sequence of *Crassula capitella* in the RPA reaction, guaranteeing the specificity of the detection method. Conventional PCR primers and RPA primers were designed within the candidate target regions using Primer Premier 5.0 software.

[0065] The design of RPA primers strictly adhered to the following principles: primer length was controlled at 30-35 bp, GC content was maintained within the range of 40%-60%, secondary structure formation and primer dimers were avoided as much as possible, high 3′ end stability was ensured, and amplicon length was controlled at 100-300 bp (generally less than 500 bp). Based on these principles, two pairs of candidate RPA primers were designed within the specific target region of *Crassula ovata*. After specificity verification and amplification efficiency comparison, both candidate primer pairs could specifically detect *Crassula ovata*, but primer sets SEQ ID NO.1 and SEQ ID NO.2 showed better band clarity and higher amplification efficiency. Therefore, SEQ ID NO.1 and SEQ ID NO.2 were ultimately selected as the optimal primer pair for the RPA detection system.

[0066] The PCR primer sequences are: F: 5′-TGTGTCATCGCTTACACTCTGTT-3′; R: 5′-GACGAAGGAAAACCAGGCAC-3′

[0067] The RPA primer sequences are: F: 5′-CGATTGTGTCATCGCTTACACTCTGTTGCC-3′ (SEQ ID NO.1) R: 5′-GACGAAGGAAAACCAGGCACTTCTCAACAC-3′ (SEQ ID NO.2)

[0068] 6. Establishment and specificity validation of the RPA detection system for oat crown rust.

[0069] Genomic DNA from oat crown rust and other common pathogens affecting cereal crops was used as templates for conventional PCR and RPA amplification reactions. ddH2O was used as a negative control, and the experiment was repeated three times.

[0070] The total volume of the PCR amplification system was 50 μL: 2 μL each of forward and reverse primers (10 μM), 2 μL template DNA (50 ng / μL), 25 μL of 2×Es Taq MasterMix (Dye) PCR mixture, and 19 μL ddH2O. The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 33 cycles: 94℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 20 s; and a final extension at 72℃ for 5 min. PCR amplification products were detected by 2% agarose gel electrophoresis, and the bands were observed using a UV gel imaging system.

[0071] The RPA amplification reaction was prepared according to the RPA reaction kit instructions, with a total reaction volume of 50 μL: 29.4 μL of buffer A, 2 μL each of forward and reverse primers (10 μM), 2 μL of template (10 ng / μL), and ddH2O added to a final volume of 47.5 μL. After thorough mixing, 2.5 μL of buffer B was added. The reaction tube was incubated in a metal bath at 39°C for 15 min. After the reaction, 50 μL of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) DNA extraction buffer was added to the reaction product. After mixing, the mixture was centrifuged at 12000 rpm for 5 min. 5 μL of the supernatant was mixed with 1 μL of 6× Loading buffer and then analyzed by 2% agarose gel electrophoresis. The amplified product bands were then observed using a UV gel imaging system.

[0072] To further optimize the RPA reaction conditions, this invention performs gradient optimization on the reaction temperature and reaction time:

[0073] Five temperature gradients were set for the reaction: 33℃, 35℃, 37℃, 39℃, and 41℃ for 30 min. The results showed that the target amplification product could be obtained at temperatures ranging from 33℃ to 41℃, but the amplification efficiency varied. The amplification product bands were brightest and showed the best results at 39℃; further increases in temperature did not result in significant changes. Therefore, 39℃ was ultimately selected as the optimal temperature. Figure 4 (a).

[0074] Six reaction time gradients were set: 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min at 39℃. The results showed that within the test range of 5-30 min, the band brightness of the RPA amplification product initially increased and then stabilized with increasing reaction time. A clear and sufficiently strong amplification band was obtained at 15 min, and further extending the reaction time did not significantly increase the band brightness. Therefore, considering both detection speed and amplification product yield, 15 min was selected as the optimal time. Figure 4 (b).

[0075] The results are as follows Figure 1 (Routine PCR) Figure 2 As shown in the RPA (Real-Range Acute Species Analysis), only the amplification system for the genomic DNA of *Crassula ovata* showed a specific target band. No amplification bands were observed in the corresponding lanes of other tested pathogens and uninoculated oat samples, which was the same as the result of the negative control (ddH2O). This indicates that the RPA detection method established in this invention has good specificity for *Crassula ovata* and can effectively distinguish it from other tested cereal pathogens.

[0076] Example 2

[0077] This embodiment provides a sensitivity test of the rapid molecular detection system for oat crown rust based on RPA.

[0078] 1. Propagate oat crown rust fungus strain for later use.

[0079] The oat variety Qingyan No. 1 was planted under the same cultivation conditions as above. Once the seedlings reached the stage where one leaf was fully expanded, a purified urediniosporium strain of fresh oat crown rust fungus was placed in a 0.5 mL centrifuge tube. An appropriate amount of electronic fluoride solution was added to prepare a spore suspension. A suitable amount of the spore suspension was then pipette-loaded and evenly applied to the upper surface of the first leaf of the oat seedlings. After inoculation, the seedlings were kept in darkness and kept moist (as above), then transferred to a cultivation greenhouse for further cultivation and collection of the inoculum (as above), resulting in a large quantity of propagated inoculum.

[0080] 2. Preparation and serial dilution of DNA template from *Crassula ovata* (oat rust fungus)

[0081] Genomic DNA template of *Crassula capitella* was extracted (as above). The extracted *Crassula capitella* genomic DNA was serially diluted at a starting concentration of 10 ng / μL, with the following concentration gradients: 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL.

[0082] 3. RPA Sensitivity Verification

[0083] Using different concentrations of *Crassula capitella* genomic DNA as templates, RPA amplification reactions were performed according to the RPA kit instructions (as above), with ddH2O as a negative control. After the reaction, the sensitivity of the *Crassula capitella* RPA detection system established in this invention was determined by 2% agarose gel electrophoresis. The experiment was repeated three times.

[0084] The results are as follows Figure 3 As shown, agarose gel electrophoresis revealed that the brightness of the amplification band gradually decreased with decreasing template DNA concentration. A target-specific amplification band of 223 bp was detected at DNA concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL. When the template concentration dropped below 1 pg / μL, the target amplification band disappeared, indicating that the detection limit of the established *Crassula ovata* RPA detection system is 1 pg / μL.

[0085] Example 3

[0086] This embodiment provides an early diagnosis and incubation period detection test for disease caused by infection with a single spore through indoor inoculation.

[0087] 1. Indoor inoculation with single urediniospores of *Crassula ovata*.

[0088] When the oat variety Qingyan No. 1 was planted and the oat seedlings were cultivated to the point where one leaf was fully flat, a 5 cm section was marked on the front of one leaf. A small amount of oat crown rust urediniospores were sprinkled on a glass slide, and a single urediniospore was picked up with an eyelash pen under a stereomicroscope and inoculated into the marked area on the leaf. After inoculation, the slide was placed in a humidified room at 10℃ in the dark for 24 hours.

[0089] 2. Sample Collection

[0090] After the dark humidification period ended, the samples were transferred to a cultivation greenhouse for further cultivation. Samples were collected every 24 hours after the humidification period ended, and the areas of the leaves were marked with lines. According to observations in this experiment, chlorosis began to appear at the inoculation site on the 7th day after inoculation, followed by sporulation. Leaf samples were collected within 10 days after inoculation for testing.

[0091] 3. Sample DNA extraction

[0092] Leaves collected at different infection times after inoculation were placed in 2 mL centrifuge tubes, two 3 mm diameter steel balls were added, and the tubes were broken up using a tissue homogenizer. Genomic DNA templates were extracted using a modified 2% CTAB method for later use.

[0093] 4. RPA testing

[0094] Using the extracted genomic DNA as a template, RPA amplification was performed (as above), and the reaction was detected by 2% agarose gel electrophoresis. ddH2O was used as a negative control, and the experiment was repeated three times.

[0095] The results are as follows Figure 5 As shown, specific amplified bands appeared from day 5 post-inoculation, and the band brightness increased with the number of days since inoculation. This result indicates that the RPA detection system for *Crassula capitella* established in this invention can sensitively detect the latent pathogen in healthy oat seedlings before sporulation symptoms appear, even in the early stage of disease development after infection with *Crassula capitella*.

[0096] In summary, this invention designed RPA primers targeting the genome sequence of *Crassula capitella*, and conducted experiments to optimize the specificity, reaction conditions, sensitivity, and latent period of single-spore infection leading to disease. An RPA detection method for *Crassula capitella* was established, demonstrating high specificity and sensitivity, effectively detecting even trace amounts of DNA. Therefore, the RPA-based molecular detection method for *Crassula capitella* proposed in this invention enables rapid detection of *Crassula capitella*, providing technical support for the rapid and efficient diagnosis of oat crown rust disease, and showing promising application prospects in field monitoring of oat crown rust pathogens.

Claims

1. A rapid molecular detection method for *Crassula capitella* based on recombinase polymerase amplification technology, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, a recombinase polymerase amplification reaction was carried out using specific primer pairs; (3) Detect the amplification products; The specific primer pair is: The forward primer is shown in SEQ ID NO.1; The reverse primer is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The recombinase polymerase amplification reaction system is as follows: 29.4 μL of Abuffer, 2 μL each of forward and reverse primers, 2 μL of template DNA, ddH2O added to a final volume of 47.5 μL, mixed well, and then 2.5 μL of B buffer is added; the concentration of both the forward and reverse primers is 10 μM.

3. The method according to claim 1 or 2, characterized in that, The temperature of the recombinase polymerase amplification reaction is 37-42℃; the reaction time is 5-20 min.

4. The method according to claim 3, characterized in that, The temperature of the recombinase polymerase amplification reaction was 39°C; the reaction time was 15 min.

5. The method according to any one of claims 1 to 3, characterized in that, The amplification products described in step (3) are detected by agarose gel electrophoresis.

6. An RPA primer pair for rapid molecular detection of *Crassula ovata*, characterized in that, The primer pair is: The forward primer is shown in SEQ ID NO.1; The reverse primer is shown in SEQ ID NO.

2.

7. The RPA primer pair according to claim 6, characterized in that, The primer pair amplifies a specific region of the translation elongation factor 1-α gene fragment in the genome of *Aureobasidium aeruginosa*, and the amplification product is 223 bp in size.

8. The application of the RPA primer pair according to claim 6 or 7 in the detection of oat crown rust.

9. The application according to claim 8, characterized in that, The test is performed on oat plant, leaf tissue, or urediniospore samples.

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